The control of flea parasites on small and large animals is not a solved problem. The presently known agents to target the flea population that infests these hosts are generally poisons that target the nervous system, such as organophosphates. These agents are notably toxic to the host as well. Accordingly, controlled doses of systemics must be used, and they are less than totally effective.
On the other hand, reagents which target flea membrane binding sites, especially those which inhabit the digestive tract, can be designed to be more flea specific. In addition, those membrane binding sites which are present in the flea midgut can provide a mechanism for direct administration to the flea through the ingestion of the blood meal.
Systemic agents against ticks, which, unlike fleas, are not insects, have been proposed where the target is the plasma membrane of the gut cells. PCT application WO 88/03929 to Biotechnology Australia et al. describes an antigen derived from a tick species or cell line that produces an immune response capable of damaging the plasma membrane of gut cells. The applicants claim that the damage is so extensive that the majority of ticks fail to survive to adult stage.
One important cell membrane receptor protein is the dimer associated with Na.sup.+ and K.sup.+ transfer across the cell membrane-(Na.sup.+ /K.sup.+)ATPase or the "sodium pump." This protein has been studied in a number of species and the genes have been cloned. Sequencing of the genes for both .alpha. and .beta. subunits shows a high degree of homology in mammalian, avian, fish, and insect (Drosophila) species. The .alpha.-subunit of the dimer is considered to be the catalytic subunit, but it appears that the .beta.-subunit may be required to position the catalytic subunit at the cell's surface.
Takeyasu, K., et al., J Biol Chem (1988) 263:4347-4354, catalog the literature relating to the retrieval of cDNAs encoding the .alpha.-subunits of piscine and mammalian sodium pumps and the .beta.-subunits of piscine, avian and mammalian pumps. This article presents information concerning the avian .alpha.-subunit which is expressed in mouse L-cells. The recombinant cells that expressed the avian .alpha.-subunits displayed high-affinity ouabain binding (ouabain is known to bind the sodium pump) and ouabain-sensitive rubidium uptake (rubidium uptake is a diagnostic for the function of the sodium pump). However, fluorescence labeling shows that mouse L-cells expressing the gene for the .alpha. subunit of the (Na.sup.+ /K.sup.+)ATPase produced the majority of the protein internal to the cells, in contrast to the almost exclusively surface distribution of the .beta.-subunit expressed in these cells (Takeyasu, K., et al., J Biol Chem (1987) 262:10733-10740).
This work was extended to Drosophila pump by the same group and described in an article by Lebovitz, R. M., et al., Embo J (1989) 8:193-102. These authors found ouabain-sensitive rubidium uptake in tissue cultures of Drosophila cells and cross-reactivity for a monoclonal antibody to the avian sodium pump .alpha.-subunit with the Drosophila pump .alpha.-subunit. The distribution in Drosophila tissues was studied by immunofluorescence microscopy and high levels of the protein were detected in the malpighian tubules, in direct flight muscles, in tubular muscles, and throughout the nervous system. The cDNA encoding the .alpha.-subunit was cloned and found to be 80% homologous to the .alpha.-subunit sequences of vertebrates. The .alpha.-subunit gene was also expressed in mouse L-cells.
The use of receptor proteins in assay systems which can be used to screen for ligands binding to such receptors has been reviewed by Strosberg, A. D., et al., in Current Opinion in Biotechnology (1991) 2:30-36. A number of formats are described which permit the use of such receptors in assays, including the expression of recombinantly-produced receptors at the surfaces of host cells. This review includes a discussion of the use of bacterial cells as hosts to provide the receptors for such screens.
The present invention is directed to flea midgut membrane cellular binding site proteins, specifically provided in recombinant form, to screen for anti-flea systemic reagents and to provide the basis for vaccine formulations.